Method for producing template-textured materials with high binding specificity and selectivity and utilization of said materials

ABSTRACT

The invention relates to a method for the production of a template-textured material by synthesis of a template-textured polymer (TTP) by performing crosslinking polymerization of functional monomers on a support in the presence of a template, which method is characterized in that a support having a thin polymer layer on the surface thereof is added with a reaction mixture consisting of polymerization initiator, template, functional monomer, crosslinker, solvent and/or buffer and, following sorption of the reaction mixture in the thin polymer layer, the polymerization is initiated and continued until the absorption capacity of the thin polymer layer for the template-textured polymer (TTP) is reached, and the template is optionally removed in a final step, the support used being selected in such a way that it cannot absorb the reaction solution. The materials of the invention are remarkable for their high binding specificity and selectivity for the template.

[0001] The invention relates to a method for the production of newtemplate-textured materials in the form of template-textured polymers(TTP) with high binding specificity and selectivity on a solid supportand their use in substance-specific separation and analytics ofmaterials.

[0002] In life sciences and biotechnology, new efficient separation andpurification strategies and detection methods for substances such asenzymes, monoclonal antibodies, recombinant proteins or smallbiomolecules are being required. Similarly, this applies to syntheticactive substances, and especially to those having a complex structureor/and relatively high molecular weight or/and limited stability.

[0003] In all these fields of use, there is an ongoing search forsubstance-specific high-performance materials, with high flexibility inadapting to the particular substances or active agents being required.Solid materials (particles, films, vessels, filters, membranes) arepreferably used to make phase separation of solid and fluid flows ofmaterial easier. In contrast to separation methods based on dissimilarphysical properties, chemical affinity to the support is a preconditionfor substance-specific separation. Substance specificity can be achievedvia interactions between biological or biomimetic ligands and receptors.For affinity separations, either specific, yet highly sensitivebiological ligands/receptors (e.g. antibodies, enzymes), or relativelyunspecific synthetic ligands (e.g. dyes, metal chelates) have been usedto date; examples are chromatography, solid-phase extraction, membraneseparation, solid-phase assays, or sensors.

[0004] Non-porous films, layers or particles having affinity ligands ontheir surfaces exhibit a low specific surface area and thus, limitedbinding capacity. Porous materials having a larger specific surface areatypically involve restricted binding capacity as a result ofdiffusion-related limitations. Analogous limitations may occur in packedparticles. Directionally permeable porous filters or membranes aretherefore particularly attractive alternative materials. Establishedmembrane processes using porous membranes, such as micro- orultrafiltration, operate according to the size exclusion principle. Theseparation of substances of similar molecular size using porousmembranes additionally requires specific (affinity) interactions withthe membrane.

[0005] The major motivation in using affinity membranes is thepossibility of directional flow onto separation-specific groups(ligands/receptors) present in the pores at high density, enabling adramatic improvement in effectiveness (less pressure drop, shorterresidence time, higher flow rate, rarely diffusion-related limitationsin pores, more rapid equilibration) as compared to analogous processesusing particles. Such affinity membranes can be used in the separationof materials, e.g. purification, preferably of proteins, but also ofmany other substances (e.g. peptides, nucleic acid derivatives,carbohydrates, or various toxins, herbicides, pesticides), and evencells. Also, decontamination of material flows is a field of use forsuch membranes, involving many applications. Furthermore, affinitymembranes provide many potential uses in analytics, e.g. in highlyselective sample accumulation, e.g. by solid-phase extraction, or in theform of a quantitative determination of a substance on an affinitymembrane, e.g. by means of ELISA.

[0006] A highly attractive alternative to biological or biomimeticaffinity ligands/receptors for the separation or analytics of materialshas been developed in recent years, involving the use of specific, yetexceedingly robust functional cavities (“molecular impressions”) insynthetic polymers, produced via molecularly texturing polymerization(G. Wulff, Angew. Chem. 107, 1995, 1958; A. G. Mayes, K. Mosbach, TrendsAnal. Chem. 16, 1997, 321; K. Haupt, K. Mosbach, Trends Biotechnol. 16,1998, 468). To this end, polymerization of monomers is effected in thepresence of template molecules (e.g. protein, nucleic acid,low-molecular weight organic substance) capable of forming a complexwith a functional monomer, which complex is relatively stable duringpolymerization. After washing out the template, the materials thusproduced are ready again to specifically bind template molecules. Thepolymers thus synthesized are referred to as template-textured polymers(TTP) or molecularly textured polymers (see FIG. 1).

[0007] Any substance having a well-defined three-dimensional morphologycan be used as a template in the synthesis of TTP. Consequently, theclasses of substances range from small molecules up to particles such asviruses, bacteria or cells. Compounds involving biological functions,such as peptides, nucleic acids or carbohydrates, are of particularinterest. The recognition of templates by TTP is based on a combinationof various factors such as reversible covalent or non-covalent bonding,electrostatic and hydrophobic interactions, hydrogen bonding andmorphological complementarity. Which of these factors will dominatedepends on template structure and properties, the functional monomer,the polymer structure, and the conditions of binding. In contrast to thecovalent approach in TTP synthesis, which requires complex syntheses oftemplate/monomer conjugates, the non-covalent approach is much moreflexible. Frequently, electrostatic interactions are suitable intemplate recognition by TTP in hydrophobic solvents. In contrast, themorphological specificity and possibly, hydrophobic interactions aremost important in template recognition in polar solvents. Preferably,TTP should be synthesized under conditions where strong, yet reversibleinteractions between polymer and template are favored. On the otherhand, a combination of much weaker bonds including hydrogen bridges andhydrophobic interactions might be favorable for large molecules (about200-1,000,000 Da). For small molecules (50-200 Da), a few stronginteractions such as ionic bonds are necessary to obtain high affinityTTP. For example, the related production of polymeric sorbents in thepresence of small organic molecules (U.S. Pat. No. 5,110,833) ormacro-molecular substances (U.S. Pat. No. 5,372,719), or the synthesisof acrylamide or agarose gels in the presence of proteins (U.S. Pat.Nos. 5,728,296, 5,756,717) have been described. Peptide- orprotein-specific sorbents produced using “surface texturing” of metalchelate structures on specifically functionalized particles (U.S. Pat.No. 5,786,428), or of carbohydrates in a plasma-polymerized layer havealso been reported. TTP membranes produced using a special “surfacetexturing” process have also been described (WO 00/07702). A significantimprovement in the synthesis of TTPs from aqueous solutions and for usein aqueous systems has been achieved by means of a special “surfacetexturing” process using special aqueous reaction solutions (patentapplication DE 198 42 641.1). In all these cases, good affinities forthe respective template have been obtained.

[0008] The use of artificial antibodies and receptors produced bymolecular texturing might involve enormous advantages, because thesestructures are much more stable compared to their natural analogues.Also, in principle, they can be synthesized for any substance (even forthose having less pronounced antigenic properties, such as smallmolecules or immunosuppressives), and their production is much easierand more cost-effective compared to corresponding biomolecules. Acrucial problem still restricting the potential uses of TTP is thatnon-specific interactions occur to a massive extent in addition to thedesired affinity of the “molecular impressions” achieved by the TTPsynthesis. In those few examples where TTP as a “plastic antibody” inassays is reported to have a remarkable template selectivity (relativelylow “cross reactivity” to similar substances), only very few templateimpressions in the TTP and thus, those having the highest affinity areutilized (K. Haupt, K. Mosbach, Trends Biotechnol. 16, 1998, 468). Ingeneral, despite the specificity of TTP for the template compared tocontrol polymers produced with no template, the latter will also bindthe template, even though to a lesser extent. Consequently, theselectivity of binding of template compared to other, structurallysimilar or different substances is limited.

[0009] The object which the invention is based upon therefore is todevelop template-textured materials having good affinity, high bindingspecificity and selectivity for the template and to provide a method ofproducing these materials.

[0010] The object of the invention is accomplished by means of a methodfor the production of a template-textured material by synthesis of atemplate-textured polymer (TTP) using a per se known crosslinkingpolymerization of functional monomers in the presence of a template on asupport, wherein, according to the invention, a support having a thinpolymer layer on the surface thereof is added with a reaction mixtureconsisting of polymerization initiator, template, functional monomer,crosslinking agent, solvent and/or buffer and, following sorption of thereaction mixture in the thin polymer layer, the polymerization isinitiated and continued until the absorption capacity of the thinpolymer layer for the template-textured polymer (TTP) is reached, andthe template is optionally removed in a final step, the support usedbeing selected in such a way that it cannot absorb the reactionsolution.

[0011] Thus, according to the invention, a support is employed whiche.g. previously has been modified on its surface with a thin polymerlayer, minimizing non-specific binding of substances, e.g. of template.It is also possible to apply said polymer layer during the synthesis ofthe support, thereby retaining the inventive two-layer structure of thesupport.

[0012] According to the invention, the supports employed are not capableof absorbing the reaction solution, while said reaction solution isabsorbed by the thin polymer layer on the support, in which layer thepolymerization takes place.

[0013] In a preferred embodiment of the invention, molded pieces made ofa hydrophobic material are used as support, while the thin polymer layersituated on the support is of hydrophilic nature.

[0014] It is in said thin polymer layer where TTP synthesis is initiatedby crosslinking polymerization of the functional monomers in thepresence of the template in aqueous or organic reaction solutions toform stable template impressions, thereby utilizing the thin polymerlayer as a matrix for TTP synthesis. Surprisingly, a heretoforeunmatched combination of TTP specificity and selectivity with minimalnon-specific binding is achieved therein. Subsequently, the syntheticreceptor structures in the form of template impressions can bindtemplate molecules or template derivatives from organic or aqueous,saline solutions in a highly specific fashion. In this way, it ispossible to use TTP materials in substance-specific methods of affinityseparation and analytics.

[0015] In another embodiment of the invention, highly crosslinkedorganic polymers or inorganic solids incapable of absorbing hydrophilicor aqueous or hydrophobic reaction solutions are used as supports, andthe thin polymer layer situated on the support can be both hydrophilicand hydrophobic in nature.

[0016] Thus, the new template-textured materials of the invention arecomprised of TTP with high binding specificity and selectivity on asolid support. Non-specific binding of substances competing withtemplate and of non-template is minimized by a previous additionalfunctionalization or coating of said support with a thin polymer layerat the surface thereof, and at the same time, the thin polymer layerserves as a matrix for the synthetic receptor structures (templateimpressions).

[0017] According to the invention, coating the support with a thinpolymer layer minimizing non-specific binding and TTP synthesis in saidpolymer layer can also be integrated into a single process step in a perse known fashion.

[0018] However, the template-textured materials of the invention arepreferably produced on a solid support of varying morphology (film,foil, plate, reaction vessel, particle, fiber, fabric, filter, membrane)with a thin polymer layer minimizing non-specific binding of template,template derivatives and other substances (see FIG. 2). As a result ofcontrolled crosslinking polymerization initiated selectively within thethin polymer layer in the presence of template, the TTP synthesis yieldsTTPs fixed covalently and/or by inclusion in the thin polymer layer(“interpenetrating network”), with template impressions over the entireexterior surface of the molded piece. Only functional groups offunctional monomer molecules complexed with template during synthesisare fixed and well-accessible on the exterior surface of the TTPmaterial. Otherwise, the thin polymer layer with its low capacity ofnon-specific binding determines the exterior surface of the TTPmaterial; in control materials synthesized with no template, this isalmost exclusively said thin, weakly binding polymer layer (cf., FIG.2). As a result of the selectivity of initiation in the thin polymerlayer, matrix structure and morphology of the support remain intact.

[0019] It is particularly preferred to produce the template-texturedmaterials of the invention on solid supports of varying morphology,which supports are functionalized by surface modification using a thinpolymer layer having minimal non-specific binding for the template,template derivatives and other substances. Thus, it is possible toachieve independent optimization of morphology or pore structure(capacity, permeability) and surface functionality (high specificity andselectivity as a result of template impressions and simultaneously,minimal non-specific binding). Particularly preferred is surfacefunctionalization of e.g. nano-, ultra- or micro-filtration membranes orfilters, wherein TTP membranes are synthesized according to theproduction method described. When filtrating through or using thematerials according to the invention, the templates or templatederivatives can also be bound from dilute solutions in the templateimpressions with high specificity. Thereafter, the templates or templatederivatives can optionally be purified and subsequently either elutedunder filtration conditions (as a concentrate) or detected by analysisand quantified directly on the support.

[0020] According to the invention, small molecules of up to 100 Da (e.g.triazine herbicides, chemical active agents, hormones, or amino acids),larger molecules of up to 1,000,000 Da (for example, peptides, proteins,nucleic acids, or carbohydrates) or particles such as viruses, bacteriaor cells are used as templates. Particularly preferred are templatescapable of interacting via ion exchange or hydrophobic binding. Usingthe present invention, ionic and electrostatic interactions, as well ashydrogen bonds can be utilized in the synthesis of TTP and thus, inmolecular recognition in aqueous systems as well. Hydrophobicinteractions may contribute in addition. Particularly in case of smallmolecules, this results in significant improvements and is also usefulfor biologically relevant molecules such as amino acids, peptides,nucleic acids, oligonucleotides, or mono- and oligosaccharides, but alsofor proteins, DNA and RNA, or polysaccharides, or conjugates thereof.The template concentrations in the reaction mixture for producing thematerials of the invention are between 0.01 and 50%.

[0021] According to the invention, polymerizable compounds includinggroups capable of interacting with templates, particularly carboxyl,sulfonyl, sulfate, phosphate, amino, or quaternary ammonium groups, aswell as derivatives thereof, also in admixture, are used as functionalmonomers. Monomers including positively or negatively charged functionalgroups (e.g. amino-functional acrylate, methacrylate or styrenederivatives, or acrylic acid, methacrylic acid,2-acryloylaminopropane-2-sulfonic acid, vinylsulfonic acid,styrenesulfonic acid, or vinylphosphonic acid) are suited for theinventive production of TTP materials. In addition, hydrophobic moietiessuch as aromatic rings, cryptands, or cyclodextrins can be incorporatedin TTP via special monomers. Monomers capable of complex formation, suchas metal chelate complexes, Schiff bases, and particular esters can alsobe used. Also, aniline and aniline derivatives including additionalfunctional groups can be employed in the inventive production of TTPmaterials. Furthermore, e.g. derivatives of phenylboronic acid capableof forming esters with diols are suitable as functional monomers.Ultimately, the functional groups in an oligomer (e.g.oligoethyleneimine) or in a polymer (e.g. agarose or polyethyleneimine)can be used in the sense of a functional monomer by way of crosslinking.The concentration of functional monomers in the reaction mixture for TTPsynthesis can be between 0.01 and 99.99%.

[0022] As is well-known, the components for a template-specific TTP arepredominantly selected according to the interactions between templateand functional monomer (formation of a supramolecular complex). Theinteractions between template and functional monomers, i.e., complexformation, can be studied and quantified using e.g. UV/VIS or NMRspectroscopy. In addition, suitable crosslinkers, solvents andinitiators are selected with the aim of “fixing” these interactions soas to be as effective and accessible for affinity interactions aspossible.

[0023] Examples of crosslinkers are bismethacrylates of ethylene glycol,diethylene glycol, triethylene glycol, or tetraethylene glycol, or otheranalogous derivatives in case of functional acrylates,N,N-methylenebisacrylamide or piperazine bisacrylamide for functionalacrylamides or methacrylamides, o-phenylenediamine for functionalaniline derivatives, or bisepoxides for agarose. The crosslinkerconcentrations in the monomer mixture are between 0 and 90%.

[0024] The solvents for polymer production can be the monomer itself,water, aqueous buffer solutions, organic solvents, or mixtures thereof.In general, the optimum type of monomer obviously depends on thetemplate structure and the conditions of polymerization.

[0025] Suitable as polymerization initiators are those compounds which(e.g. peroxides, azo compounds) upon excitation either undergo cleavageof bonds, or (in case of e.g. H-abstracting agents), by way ofbimolecular reaction with other molecules/structures, will form freeradicals capable of initiating polymerization. Particularly suited arephotoinitiators such as benzophenone and derivatives thereof. Dependingon polymerization conditions and composition, textured polymers can beproduced which have the desired density, porosity, crosslinking density,and consistency. This procedure is well-known to those skilled in theart.

[0026] A variety of different support forms such as films, foils,plates, reaction vessels, microtiter plates, (micro and nano)particles,fibers, hollow fibers, fabrics, fleeces, filters, or membranes made ofvarious organic or inorganic materials can be used in the inventiveproduction of template-textured materials. Organic materials arepolymers such as polypropylene, polyethylene, polystyrene, polysulfone,polyamides, polyesters, polycarbonate, polyacrylonitrile,poly(vinylidene fluoride), polytetrafluoroethylene, polyacrylates,polyacrylamides, cellulose, amylose, agarose, as well as derivatives,copolymers or blends thereof. Inorganic materials are e.g. glasses,silicates, ceramics, or metals or composites thereof, also includingorganic polymers. The supports can be non-porous or porous. Particularlypreferred with regard to production and use of the new template-texturedmaterials are supports in the form of polymer membranes which can beproduced by means of processes such as precipitant- ortemperature-induced phase inversion with a variety of pore structuresand with the desired mechanical or other properties. The membranespreferably have symmetrical, but also asymmetrical pore structures and apore size ranging between a few nm and 10 μm, preferably from 100 nm to5 μm. In this way, the optimum porous matrix membranes (supports) can beselected for the desired separation and analytic processes. According tothe invention, however, membranes (supports) having a thin polymer layeron the surface thereof are particularly preferred. They are eithercommercially available (e.g. hydrophilized PVDF membranes) or can beproduced according to principally well-known procedures (e.g. U.S. Pat.No. 4,618,533).

[0027] The inventive production of the template-textured materials isperformed using a reaction mixture including at least template andfunctional monomer. The reaction proceeds so as to retain the complex oftemplate and functional monomer and thus, the fundamental preconditionas to formation of synthetic receptors (template impressions) is given.According to the invention, neither stability nor pore structure of thesupport will be impaired.

[0028] By selecting the polymer layer on the support, it is possible tominimize non-specific interactions of structurally similar or differentsubstances with TTP, in addition to achieving high affinity of thetemplate impressions. Thus, the specificity of TTP compared to thecontrol, as well as the selectivity of TTP for the template compared toother substances are significantly increased (cf., FIG. 2).

[0029] The following production procedure is particularly suited for thesynthesis of TTP materials of the invention having high bindingspecificity and selectivity:

[0030] 1. a) Selecting a support with a thin polymer layer on itssurface, which minimizes non-specific binding of substances, e.g.template, on the support, or

[0031] b) production of a support (during synthesis or processing of thesupport material or by surface modification of the support) with a thinpolymer layer on its surface, which minimizes non-specific binding ofsubstances, e.g. template, on the support;

[0032] 2. Coating the support with the major portion of polymerizationinitiator—accumulation of initiator in the thin polymer layer;

[0033] 3. Coating the support with the reaction mixture (template,functional monomer, crosslinker, solvent and/or buffer, residual portionof initiator)—sorption in the thin polymer layer;

[0034] 4. Initiating the polymerization and continuing so as to reachthe absorption capacity of the thin polymer layer—preferred formation ofstarter free radicals and polymerization in the thin polymer layer;

[0035] 5. Optionally extraction of non-reacted reactants, solublehomopolymer and template.

[0036] In another embodiment of the invention, the polymerizationinitiator can also be added completely in step 2.

[0037] A substance forming free radicals or other starter species forpolymerization upon physical or chemical excitation is employed asinitiator. Functionalization can be based on the action of the thinpolymer layer as co-initiator, i.e., all those polymers from which freeradicals or other species capable of initiating a graft copolymerizationcan be generated by means of initiators can be modified in this way.

[0038] Photochemical initiation of a heterogeneous graftcopolymerization (of e.g. functional acrylates) is particularlypreferred in the inventive production of template-textured materials.This is done by using a photoinitiator, particularly of an H abstractiontype, and selective UV irradiation of the photoinitiator in step 4. Suchpolymerization may proceed at low temperatures particularly favorable inTTP synthesis (T≦25° C.) where impairment of the supramolecular complexof template and functional monomer is low.

[0039] Selectively initiated chemical grafting or crosslinking ofpolymers (e.g. synthesis of polyaniline derivatives) on the supporthaving the thin polymer layer is also suitable in the inventiveproduction of template-textured materials. Also, the synthesis of aninterpenetrating network by selectively initiated polymerization of theTTP in the thin polymer layer and anchoring by interpenetration and/orintertwining with no chemical reaction between the two polymers mayresult in TTP having high specificity and selectivity.

[0040] As is well-known, TTP syntheses can be effected using surfacefunctionalization from aqueous or organic solvents. The degree offunctionalization and thus, the surface coverage of the support with TTPcan be controlled via initiation and polymerization conditions. Ifnecessary, blocking of pores in the support can also be minimized inthis way. Owing to the large number of variants, the whole spectrum ofmethods established for TTP synthesis is also applicable to surfacefunctionalization of the particular support materials used according tothe invention.

[0041] In the production of TTP, the binding specificity and capacity ofthe template-textured polymer for the template and template-likesubstances can be increased by adding a salt to the reaction solution(in step 3), e.g. in the form of a buffer.

[0042] To wash out the template from the TTP, it is possible to use e.g.an acid interfering with the electrostatic interactions, a salt solutionhaving an ionic strength sufficient for dissociation, or a solventhaving a different polarity. In this way, the binding sitescomplementary to the template structure return to the blank state in thepores or/and on the surface of the material of the invention. However,the materials of the invention can also be used with bound template.

[0043] Characterization of the properties of the materials according tothe invention is effected in a basically well-known manner usingwell-established methods, e.g. SEM investigations, measurement of thespecific surface area (BET isotherm), FT-IR-ATR spectroscopy, analyticsof functional groups using photometric or fluorimetric methods, contactangle measurement, as well as permeability measurement.

[0044] Characterization of the properties of the materials according tothe invention is effected in a basically well-known manner using staticand dynamic sorption experiments on the template or other structurallysimilar or different substances. In particular, the binding capacitiesof the materials of the invention for template as a function of the TTPstructure of the material and the test conditions (concentration,residence time, amount and volume of substance applied, rinsingconditions; particularly, also in mixtures with other substances) areessential in view of the diverse uses of the materials according to theinvention.

[0045] When applied on or filtrated through template-textured materialsof the invention, the templates or template derivatives are bound in thetemplate impressions with high specificity, even from a high dilution.Thereafter, the templates or template derivatives can be purified bywashing and subsequently either detected directly on the support oreluted under filtration conditions (in the form of a concentrate) (seeFIG. 3). Hence, the TTP materials of the invention with high bindingspecificity and selectivity enable effective, substance-specificseparation of materials or/and analytic determinations from organic oraqueous solutions.

[0046] The invention is also directed to the template-textured materialsproduced using the method of the invention and to the use thereof.

[0047] According to the invention, the new template-textured materialsare used in the separation of materials and/or in the analytics of fluidor gaseous mixtures of substances, which procedures are based on thespecific binding of templates or template derivatives during perfusionor diffusion through template-textured polymers or when applied ontemplate-textured polymers.

[0048] The TTP materials of the invention provide the following uses,although it is not intended to restrict the possible uses to theseconcrete cases:

[0049] 1. Separation: use in solid-phase extraction, chromatography,electrophoresis, membrane separation or controlled release of activeagents—filtration (perfusion) or affinity filtration, diffusion(dialysis) or electrodiffusion (electrodialysis) of solutions or gaseousmixtures through the template-textured materials of the invention, orsorption thereon to effect concentration, purification, separation orsubsequent analytical determination of substances;

[0050] 2. Analytics: use as test strip, blotting membrane or sensitivelayer in assays in reaction vessels or microtiter plates (e.g.qualitative or quantitative determinations or active agentdetection/screening)—applying solutions or gaseous mixtures on TTPmaterials or sorption on TTP materials;

[0051] 3. Sensor technique: use as receptor or/and transducer; use inoptionally continuous analytical determination of substances;

[0052] 4. Catalysis: use of TTP as receptor and/or catalytically activecenter; use in synthesis, purification, separation or analyticaldetermination of substances.

[0053] Suitable classes of substances for the above-mentioned uses rangefrom small molecules of up to 100 Da (e.g. triazine herbicides orhormones) up to particles such as viruses, bacteria or cells. Inparticular, they are biologically relevant molecules (active substances)such as amino acids, peptides, nucleic acids, oligonucleotides, or mono-and oligosaccharides, but also proteins, DNA and RNA, or polysaccharidesor conjugates thereof.

[0054] Providing a combination of good affinity, high specificity andselectivity, as well as low non-specific binding, the materials of theinvention involve the advantage of enabling new, highly efficientmethods of substance-specific separation and analytics of materials. Inparticular, the TTP materials of the invention can also be produced fromaqueous reaction mixtures. Thus, the method according to the inventioncan also be applied to biomolecules, with the activity thereof beingretained. Hence, the new TTP materials can also be used in theseparation and analytics of materials from/in aqueous solutions andthus, particularly in life science and biotechnology.

[0055] Without intending to be limiting, the invention will beillustrated in more detail with reference to the embodiments andfigures.

EXAMPLES Example 1 Poly(Vinylidene Fluoride) Membranes Template-Texturedfor Terbumeton (2-t-butylamino-4-ethyl-6-methoxy-1,3,5-triazine)

[0056] a) Variation of Functional Monomer

[0057] Round samples (4.9 cm²) of hydrophilized PVDF membranes (poresize 0.22 μm; “Hydrophilisierte Durapore”; Millipore GmbH, Eschborn,Germany) are extracted with acetone and methanol, dried and weighed.Thereafter, the samples are immersed in a 150 mM solution of BP(photoinitiator) in acetone for 5 min and then dried under vacuum.Subsequently, the membranes in Petri dishes are covered with a layer ofreaction solution consisting of 10 mM Terbumeton (template; PESTANAL;Riedel de Haën GmbH & Co. K G, Seelze, Germany), 50 mM AA, MAA and AMPS,respectively (functional monomer; Sigma-Aldrich), 300 mM MBAA(crosslinker; Sigma-Aldrich), and 5 mM BP in methanol. The Petri dish iscovered with a glass plate (Tief UV filter, λ>310 nm). After 10 min,irradiation is effected on a UV dryer (Beltron GmbH) at half power for atotal of 10 min (10 passages through the irradiation zone).Subsequently, the membranes are extracted thoroughly in a Soxhletapparatus for 2 hours and washed with water, 50 mM hydrochloric acid,water, and methanol. Thereafter, this is dried, and the degree ofmodification (DM, based on exterior membrane surface) is determined bygravimetry.

[0058] Preparations under the above conditions are also carried out withnon-hydrophilized PVDF membranes (pore size 0.22 μm; “HydrophobeDurapore”; Millipore GmbH, Eschborn, Germany).

[0059] b) Variation of the Functional Monomer Concentration

[0060] Preparation is as described above; however, reaction solutionsare used consisting of 10 mM Terbumeton, from 0 to 60 mM AMPS, 300 mMMBAA and 5 mM BP in methanol.

[0061] c) Variation of Crosslinker Concentration

[0062] Preparation is as described above; however, reaction solutionsare used consisting of 10 mM Terbumeton, 50 mM AMPS, from 200 to 350 mMMBAA and 5 mM BP in methanol. Non-template-textured control samples areprepared in a procedure analogous to that for TTP membranes, but with notemplate. All results are summarized in Table 1. TABLE 1 Degree ofmodification (DM, in μg/cm²) for PVDF membranes following syntheses with(TTP) or without (blank) the Terbumeton template (cf., Example 1) a)Functional monomer AA MAA AMPS hydrophilized TTP1a 380 360 340 DuraporeBlank1a 320 310 340 hydrophobic TTP1k — — 0 Durapore Blank1k — — 0 b)AMPS functional monomer 0 mM 20 mM 40 mM 50 mM 60 mM 1 TTP1b 350 370 380340 410 Blank1b 380 400 380 340 420 c) MBAA 200 225 250 275 300 325 350crosslinker mM mM mM mM mM mM mM 2 TTP1c 350 300 310 370 340 340 360Blank1c 390 350 360 360 340 330 320

[0063] Similar DM values are obtained for TTP and blank materials underanalogous conditions. A significant increase is observed for TTP1b andBlank1b from 60 mM AMPS on, indicating modification beyond the capacityof the thin hydrophilic polymer layer (cf., FIG. 2). Investigations ofstructure using SEM, BET, ATR-IR, as well as functional group assaysestablish modification with a thin functional polyacrylate layer;differences in composition between TTP and blank materials cannot bedetected.

Example 2 Use of Poly(Vinylidene Fluoride) Membranes Template-Texturedfor Terbumeton in Substance-Specific Membrane Solid-Phase Extraction

[0064] Round samples (4.9 cm²) of modified membranes as in Example 1 aremounted in a steel filter holder having a Luer-Lock connection(effective membrane area 3.8 cm²; Schleicher & Schuell GmbH, Dassel,Germany). 10 ml of a 10⁻⁵ M solution of herbicide (Terbumeton, Atrazine,Desmetryn, Terbutylazine, Metribuzin; PESTANAL; Riedel de Haën GmbH &Co. K G, Seelze, Germany) in water is filtrated quantitatively from asyringe through the membrane at a rate of 10 ml/min. Subsequently, boththe filtrate and 10 ml of crude solution are extracted using 10 ml ofchloroform each time. Both herbicide concentrations are then determinedquantitatively using gas chromatography (HP5MS separation column;Hewlett Packard GC System HP 6890 including HP 5973 mass-selectivedetector); the amount bound in the membrane is calculated from theconcentrations of crude solution and filtrate.

[0065] Summaries of the results are given in FIG. 4 for the variation offunctional monomer (see Example 1a), in FIG. 5 for the variation offunctional monomer concentration (see Example 1b), and in FIG. 6 for thevariation of crosslinker concentration (see Example 1c).

[0066] At low absolute values for non-specific binding (“background”; <2μmol/cm²), the sorption values for TTP materials are significantlyhigher than those for the corresponding blank materials; AMPS, beingthat functional monomer with the most intense complex formation withtemplate as compared to AA and MAA, results in TTP with maximum affinity(see FIG. 4).

[0067] At relatively low absolute values for non-specific binding(“background”; <5 μmol/cm²), the sorption values for TTP materials willnot be significantly higher than those for the corresponding blankmaterials before an AMPS concentration of 40 mM is reached; excessivelyhigh AMPS concentrations result in excessively high DM values (cf.,Table 1b) and thus, in a significant increase of non-specific sorption(see FIG. 5). It is only at an optimum crosslinker concentration thatlow absolute values for non-specific binding (“background”; <5 μmol/cm²)and higher sorption values for TTP materials compared to thecorresponding blank materials are obtained with the functional (cationexchange) AMPS monomer (see FIG. 6).

Example 3 Poly(Vinylidene Fluoride) Membranes Template-Textured forDesmetryn (2-isopropylamino-4-methylamino-6-methylthio-1,3,5-triazine)

[0068] A round sample (46 cm²) of a hydrophilized PVDF membrane (cf.,Example 1) is extracted with chloroform and methanol, dried and weighed.Thereafter, the membrane is immersed in a 100 mM solution of BP inmethanol for 30 min. Subsequently, the membrane, the pores of whichstill being filled with BP solution, is covered in a Petri dish (d=10cm) with a layer of reaction solution consisting of 10 mM Desmetryn(template), 50 mM AMPS (functional monomer), 100 mM MBAA (crosslinker),and 0.1 mM BP in water. The Petri dish is covered with a glass plate(Tief UV filter, λ>310 nm). After 30 min, irradiation is effected on aUV dryer (Beltron GmbH) at half power for a total of 10 min (10 passagesthrough the irradiation zone). Subsequently, the membrane is washedthoroughly with methanol, water, 50 mM hydrochloric acid, water, andmethanol again. Thereafter, this is dried, and the degree ofmodification (DM, based on exterior membrane surface) is determined bygravimetry. Non-template-textured control samples are prepared accordingto an analogous protocol, but with no template. The results forpreparation conditions varied according to the above-mentioned generalprotocol are shown in Table 2.

[0069] TTP and blank materials can also be synthesized from aqueoussolutions, and similar DM values are obtained under analogous conditionseach time.

Example 4 Use of Poly(Vinylidene Fluoride) Membranes Template-Texturedfor Desmetryn in Substance-Specific Membrane Solid-Phase Extraction

[0070] Round samples (4.9 cm²) of membranes modified according toExample 3 are mounted in a steel filter holder having a Luer-Lockconnection (effective membrane area 3.8 cm²; Schleicher & Schuell GmbH,Dassel, Germany). 10 ml of a 10⁻⁵ M solution of herbicide (Desmetryn) inwater or 50 mM sodium phosphate buffer (pH=5.0) is filtratedquantitatively from a syringe through the membrane at a rate of 10ml/min. Subsequently, both the filtrate and 10 ml of crude solution areextracted using 10 ml of chloroform each time. Both herbicideconcentrations are then determined quantitatively using gaschromatography (HP5MS separation column; Hewlett Packard GC System HP6890 including HP 5973 mass-selective detector); the amount bound in themembrane is calculated from the concentrations of crude solution andfiltrate, obtaining the herbicide sorption values illustrated in Table2.

[0071] The results of binding different herbicides (see FIG. 7) show aremarkable substance specificity which is unexpected when compared tothe prior art: it is only the TTP material that exhibits significantbinding for a triazine herbicide, and said binding is obtainedexclusively for the Terbumeton template used in synthesis, not for thestructurally highly similar triazine herbicides Metribuzin, Desmetryn,Atrazine, and Terbutryn.

[0072] With small absolute values for non-specific binding(“background”; <5 μmol/cm²), the sorption values for TTP materials aresignificantly higher than those for the corresponding blank materials.In particular, this also applies to TTP materials synthesized by varyingpH and salt; the latter can bind template specifically from buffersolutions as well.

[0073] The TTP-bound herbicide can be eluted from the membrane byaltering the pH or increasing the salt concentration. Thus, using 10 mlof a 100 mM solution of sodium chloride in water, 89% is eluted from theTTP 2/1 membrane after Desmetryn sorption from water (cf., Table 2). Inan analogous fashion, with a sorption of 100% and a retrieval of 90%,the herbicide can be accumulated by 1000 fold from a 1×10⁻⁹ M solution,i.e., substance-specific solid-phase extraction can be used both inpurification and concentration. The TTP membranes can be used repeatedlyafter a simple regeneration, without loss of specificity and capacity.TABLE 2 Degrees of modification (DM; cf., Example 3) and Desmetrynsorption (from water or buffer, pH 5.0) in filtration (cf., Example 4)for hydrophilized PVDF membranes following syntheses with (TTP) andwithout (blank) the Desmetryn template Functional- Reaction izationsolution: DM Sorption n PVDF-h PH salt (nM) (ng/cm²) S (μmol/cm²)/% TTP2/1 1.5 1 305 Water 17.2/65.4  Blank 2/1 1.5 1 285 Water 2.0/7.6  TTP2/1 1.5 1 305 Buffer 3.0/11.4 Blank 2/1 1.5 1 285 Buffer 3.2/12.2 TTP2/2 1.5 50 310 Buffer 13.9/52.9  Blank 2/2 1.5 50 290 Buffer 4.2/16.0TTP 2/3 2.1 50 320 Buffer 9.9/37.6 Blank 2/3 2.1 50 305 Buffer 3.8/14.4

Example 5 Production of a Polypropylene Membrane Support Having a ThinHydrophilic Crosslinked Polymer Layer (PP-h)

[0074] A PP membrane (39 cm²; pore size 0.2 μm; Accurel PP 2E HF;Akzo-Nobel AG, Wuppertal, Germany) is equilibrated with a 100 mMsolution of BP in acetone with agitation for 2 hours. The membrane istaken out of the solution and, following removal of the solutionadhering to the outside thereof, immediately covered in a Petri dishwith a layer of a reaction solution consisting of 75 g/l 2-hydroxypropylmethacrylate and 7.5 g/l tetraethylene glycol bismethacrylate (each fromRöhm GmbH, Darmstadt, Germany) in water saturated with BP. The Petridish is covered with a glass plate (Tief UV filter, λ>310 nm). After 5min, irradiation is effected on a UV dryer (Beltron GmbH) at half powerfor a total of 10 min (10 passages through the irradiation zone).Subsequently, the membrane is extracted in a Soxhlet apparatus withwater for 2 hours and subsequently washed with acetone and methanol.Thereafter, this is dried, and the degree of modification is determinedby gravimetry: DM (PP-h)=320 μg/cm². Given the specific surface area ofthe membrane material (PP) of 17.5 m²/g and assuming a density of thegraft polymer of 1 g/cm³, this DM value corresponds to a layer thicknessof the hydrophilic crosslinked polymer layer of about 10 nm.

[0075] Surface-modified membranes having a thin polymer layer with lowernon-specific binding due to hydrophilicity can be produced as specialsupports for the inventive TTP synthesis.

Example 6 Polypropylene Membrane Template-Textured for Terbumeton andIts Use in Membrane Solid-Phase Extraction

[0076] A PP membrane (39 cm²; PP-h) modified according to Example 5 isimmersed in a 150 mM solution of BP in acetone for 5 min and then driedunder vacuum. Subsequently, the membrane in a Petri dish is covered witha layer of reaction solution consisting of 10 mM Terbumeton (template),50 mM AMPS (functional monomer), 300 mM MBAA (crosslinker), and 5 mM BPin methanol. The Petri dish is covered with a glass plate (Tief UVfilter, λ>310 nm). After 10 min, irradiation is effected on a UV dryer(Beltron GmbH) at half power for a total of 10 min (10 passages throughthe irradiation zone). Subsequently, the membrane is extractedthoroughly in a Soxhlet apparatus for 2 hours and washed with water, 50mM hydrochloric acid, water, and methanol. Thereafter, this is dried,and the degree of modification is determined by gravimetry (see Table3). A non-template-textured control sample is prepared according to ananalogous protocol, but with no template. Following functionalcharacterization as described in Example 2, the values illustrated inTable 3 are obtained for herbicide sorption. TABLE 3 Degrees ofmodification (DM) and Terbumeton sorption from water in filtration forhydrophilized PP membranes following syntheses with (TTP) and without(blank) the Terbumeton template Functionalization Sorption PP-h DM(μg/cm²) n (μmol/cm²)/% TTP 3/1 345 11.1/42.2 Blank 3/1 325 2.1/8.0 PP,non-modified — 22.4/85.0

[0077] With low absolute values for non-specific binding (“background”;<3 μmol/cm²; cf., “PP, non-modified”) which is due to previous coatingwith a thin, crosslinked, hydrophilic polymer layer (cf., Example 5),the sorption values for TTP materials are significantly higher thanthose for the corresponding blank materials.

Example 7 Polypropylene Membrane Template-Textured for Desmetryn and ItsUse in Membrane Solid-Phase Extraction

[0078] A PP membrane (39 cm²; PP-h) modified according to Example 5 isimmersed in a 100 mM solution of BP in methanol for 30 min.Subsequently, the membrane, the pores of which still being filled withBP solution, is covered in a Petri dish (d=10 cm) with a layer ofreaction solution consisting of 10 mM Desmetryn (template), 50 mM AMPS(functional monomer), 100 mM MBAA (crosslinker), and 0.1 mM BP in water.The Petri dish is covered with a glass plate (Tief UV filter, λ>310 nm).After 30 min, irradiation is effected on a UV dryer (Beltron GmbH) athalf power for a total of 10 min (10 passages through the irradiationzone). Subsequently, the membrane is washed thoroughly with methanol,water, 50 mM hydrochloric acid, water, and methanol again. Thereafter,this is dried, and the degree of modification is determined bygravimetry. A non-template-textured control sample is prepared accordingto an analogous protocol, but with no template (see Table 4). Followingfunctional characterization as described in Example 4, the valuesillustrated in Table 4 are obtained for herbicide sorption. TABLE 4Degrees of modification (DM) and Desmetryn sorption from water infiltration (cf., Example 4) for hydrophilized PP membranes followingsyntheses with (TTP) and without (blank) the Desmetryn templateFunctionalization Sorption PP-h DM (μg/cm²) n (μmol/cm²)/% TTP 4/1 3159.9/37.6 Blank 4/1 310 1.2/4.6  PP, non-modified — 11.6/44.0 

[0079] With low absolute values for non-specific binding (“background”;<2 μmol/cm²; cf., “PP, non-modified”) which is due to previous coatingwith a thin, crosslinked, hydrophilic polymer layer (cf., Example 5),the sorption values for TTP materials are significantly higher thanthose for the corresponding blank materials even upon synthesis fromaqueous reaction mixtures.

[0080] The very high specificity of the TTP materials according to theinvention (as compared to the control samples) and the very hightemplate selectivity (in relation to other structurally highly similartriazine herbicides), as well as the flexibility of the syntheticprocess compared to the prior art will be illustrated in Table 5. TABLE5 Comparison of the TTP materials of the invention with prior artmaterials (synthesis by surface functionalization viagraft-photopolymerization: functional monomer: AMPS, stoichiometryrelative to template: 5:1, crosslinker: MBAA) relating to the use inmembrane solid-phase extraction (selectivity: in all cases comparisonbetween sorption for Terbumeton and Desmetryn) Membrane solid-phaseextraction Non- specific Selectivity sorption Speci- template/ Synthesisn ficity non- Template Solvent (μmol/cm²) TTP/Blank template PP^(#)Desmetryn Water 15.0 1.6 1.08 PVDF Desmetryn Water Synthesis notpossible PP-h^(a) Desmetryn Water 1.2 8.2 n.d. PVDF- Desmetryn Water 2.08.6 6.9 h^(b) PP^(#) Terbumeton MeOH Synthesis not possible PVDFTerbumeton MeOH Synthesis not possible PVDF- Terbumeton MeOH 2.1 5.3n.d. h^(c) PVDF- Terbumeton MeOH 0.5 22.0 10.8 h^(d)

Example 8 Polyaniline-Modified Polypropylene Membrane Textured forMetribuzin (4-amino-3-methylthio-6-tert-butylamino-1,2,4-triazin-5-one)and Its Use in Membrane Solid-Phase Extraction

[0081] A PP membrane (25 cm²) modified according to Example 5 is placedin 10 ml of a 200 mM solution of aniline hydrochloride and 50 mMMetribuzin in water. After 10 min, 10 ml of a 100 mM solution ofammonium peroxodisulfate oxidant is added and reacted for 15 min withagitation (300 rpm). Subsequently, the membrane is washed thoroughlywith 10 mM hydrochloric acid, water, and methanol. Thereafter, this isdried, and the degree of modification is determined by gravimetry. Anon-template-textured control sample is prepared according to ananalogous protocol, but with no template (see Table 6). Followingfunctional characterization with Metribuzin in the way as described inExample 2, the values illustrated in Table 4 are obtained for herbicidesorption.

[0082] Chemical initiation of grafting and crosslinking of a functionalpolymer on a special support with a thin polymer layer is also suitableas an alternative synthesis of the TTP materials according to theinvention. TABLE 6 Degrees of modification (DM) and Metribuzin sorptionfrom water in filtration (cf., Example 4) for hydrophilized PP membranesfollowing syntheses with (TTP) and without (blank) the Metribuzintemplate Functionalization Sorption PP-h DM (μg/cm²) n (μmol/cm²)/% TTP5/1 210 7.9/30.0 Blank 5/1 230 4.1/15.6

Example 9 Modification of Polypropylene Microtiter Plates with a ThinHydrophilic Crosslinked Polymer Layer (PP-h)

[0083] 100 μl of a 100 mM solution of BP in acetone is pipetted in eachwell of a 96 well microtiter plate made of PP (flat bottom; CorningCostar Germany, Bodenheim), and the plate is sealed. After 2 hours, thesolution is pipetted out, the wells are rinsed with 120 μl of a 1 mMsolution of BP in acetone for 10 seconds and subsequently air-dried for15 minutes. Thereafter, 100 μl of a reaction solution consisting of 75g/l 2-hydroxypropyl methacrylate (Röhm) and 7.5 g/l tetraethylene glycolbismethacrylate (Röhm) in water saturated with BP is pipetted in eachwell. The microtiter plate is covered with a glass plate (Tief UVfilter, λ>310 nm). After 5 min, irradiation is effected on a UV dryer(Beltron GmbH) at half power for a total of 10 min (10 passages throughthe irradiation zone). Subsequently, the microtiter plate is washed withhot water first, and then with acetone and methanol.

[0084] Surface-modified microtiter plates with a thin polymer layerhaving low non-specific binding can be produced as special supports forTTP synthesis according to the invention.

Example 10 Microtiter Plate Textured for Atrazine (MTP-TTP)

[0085] 100 μl of a 100 mM solution of BP in acetone is pipetted in eachwell of a 96 well microtiter plate modified according to Example 9(PP-h), and the plate is sealed. After 2 hours, the solution is pipettedout, the wells are rinsed with 120 μl of a 1 mM solution of BP inacetone for 10 seconds and subsequently air-dried for 15 minutes.Thereafter, 100 μl of a reaction solution consisting of 10 mM Atrazine(template), 50 mM AMPS (functional monomer), 300 mM MBAA (crosslinker),and 5 mM BP in methanol is pipetted in each well. The microtiter plateis covered with a glass plate (Tief UV filter, λ>310 nm). After 30 min,irradiation is effected on a UV dryer (Beltron GmbH) at half power for atotal of 10 min (10 passages through the irradiation zone).Subsequently, the microtiter plate is washed with hot water first, andthen with 50 mM hydrochloric acid, water and methanol. A controlpreparation (MTP-Blank) is produced in an analogous fashion, but with noAtrazine.

Example 11 Replacement of Biological Receptors (in this Case: Antibodiesto Atrazine) in Microtiter Plate Assays by TTP Surfaces

[0086] The surfaces of the PP microtiter plate wells (MTP-TTP) obtainedin Example 10 exhibit properties of artificial antibodies to Atrazine,and this can be utilized in a competitive triazine assay:

[0087] 50 μl of a solution of herbicide (Atrazine or Metribuzin) atconcentrations of from 10⁻⁷ to 10⁻⁴ M in water and then 50 μl ofAtrazine-peroxidase conjugate solution (from the PESTANAL Atrazine ELISAKit; Riedel de Haën) are pipetted into different TTP Wells and incubatedwith agitation at room temperature for 2 hours. Subsequently, this iswashed, developed and quenched according to the protocol of thecommercial assay (see above). The absorbances at 450 nm are measured ina microtiter plate reader; measurements made in wells whereinmodification has been effected without template (MTP-Blank) anddeterminations using another herbicide are used as controls (see Table7). TABLE 7 Results of a competitive assay for Atrazine usingAtrazine-peroxidase conjugate and developer solutions (POD Assay) from acommercial Atrazine ELISA kit with Atrazine-textured (MTP-TTP) andcontrol wells (MTP-Blank) of a PP microtiter plate Absorbance (450 nm)Atrazine concentration 10⁻⁷ 10⁻⁶ 10⁻⁵ 10⁻⁴ MTP-TTP 0.75 0.69 0.61 0.55Metribuzin concentration (nM) 0.78 0.73 0.76 0.69 MTP-TTP 10⁻⁷ 10⁻⁶ 10⁻⁵10⁻⁴ MTP-Blank 0.78 0.77 0.74 0.72

[0088] In an assay relating to the analytics of herbicides in an ELISAformate and according to an established ELISA protocol, the new TTPmaterials show pronounced specificity (TTP vs. blank) and selectivity(Atrazine template vs. Metribuzin non-template).

Example 12 Microtiter Plate Textured for Peroxidase (MTP-TTP)

[0089] In the wells of a 96 well microtiter plate modified according toExample 9 (PP-h), aniline is polymerized according to the followingprotocol: 20 μl of ammonium peroxodisulfate (250 mM in water) ispipetted into 30 μl of a solution of aniline hydrochloride (720 mM) andhorseradish peroxidase (1.67 mg/ml) in water, and this is mixedthoroughly and reacted at room temperature with agitation for 2 hours.Thereafter, this is washed thoroughly with water and subsequently with10 mM sodium phosphate buffer (pH=7.5). A control preparation (HP-Blank)is produced in an analogous fashion, but with no peroxidase.

Example 13 Replacement of Biological Receptors (in this Case: Antibodiesto Peroxidase) in Microtiter Plate Assays by TTP Surfaces

[0090] A microtiter plate modified according to Example 12 exhibitsproperties of artificial antibodies to peroxidase.

[0091] To demonstrate the affinity of TTP surfaces for the template,horseradish peroxidase is adsorbed from a solution at a concentration of1 g/l with agitation at room temperature for 2 hours. Thereafter, thisis washed thoroughly with 10 mM sodium phosphate buffer (pH=7.5),followed by measuring the POD activity using the developer solutionsfrom the PESTANAL Atrazine ELISA Kit (Riedel de Haën) according to theprotocols of the kit. Significantly higher absorbance values (450 nm)for the TTP surface (MTP-TTP/POD: 0.27±0.08) compared to thenon-textured control sample (MTP-Blank/POD: 0.12±0.06) indicatepreferred binding of peroxidase to the synthetic receptor structures.

Example 14 Production of Microparticle Supports with a Thin HydrophilicCrosslinked Polymer Layer (MP-h)

[0092] A suspension (100 mg/ml; 10 ml) of microparticles (diameter: 3μm, styrene-MSA copolymer core with hydroxy-functional surface;microcaps GmbH, Rostock, Germany) in water is added with 10 ml of afreshly prepared solution of 50 g/l 2-hydroxypropyl methacrylate and 5g/l tetraethylene glycol bismethacrylate in 0.08 M aqueous nitric acidwith vigorous stirring, and this is purged intensively with nitrogen andheated to 50° C. Thereafter, 4×10⁻³ M ceric ammonium nitrate is added,and the polymerization is performed for 2 hours at 50° C. under nitrogenand with vigorous stirring. Thereafter, 80 ml of water is added, andthis is centrifuged, washed with methanol, resuspended in water, andsubsequently washed thoroughly with water (dialysis against water).

[0093] Surface-modified microparticles with a thin polymer layer havinglow non-specific binding due to hydrophilicity can be produced asspecial supports for the TTP synthesis according to the invention.

Example 15 Microparticles Textured for Atrazine (MP-TTP)

[0094] A suspension (100 mg/ml; 5 ml) of microparticles modifiedaccording to Example 14 in methanol (MP-h) is added with 5 ml of areaction solution consisting of 20 mM Atrazine (template), 100 mM AMPS(functional monomer), 600 mM MBAA (crosslinker), and 20 mM BP inmethanol. The suspension is placed in a flat dish on a stirring plate,covered tightly with a glass plate, purged with nitrogen, and stirredvigorously. After 30 min, irradiation is effected for 15 min, using a UVlamp (UVA Spot 2000 with Tief UV filter H2; Dr. Hönle GmbH, Planegg,Germany). Thereafter, 40 ml of water is added; this is centrifuged andsubsequently washed with hot water first, then with 50 mM hydrochloricacid, water and methanol (each time completed by centrifuging); this isfollowed by resuspending and thorough washing with water (dialysis). Acontrol preparation (MP-Blank) is produced in an analogous fashion, butwith no Atrazine.

Example 16 Replacement of Biological Receptors (in this Case: Antibodiesto Atrazine) in Microparticle Assays by TTP Surfaces

[0095] The surfaces of the microparticles obtained in Example 15(MP-TTP) exhibit properties of artificial antibodies to Atrazine, andthis can be utilized in a competitive triazine assay:

[0096] 50 μl of a solution of herbicide (Atrazine or Metribuzin) atconcentrations of 10⁻⁵ M in water and then 50 μl of Atrazine-peroxidaseconjugate solution (from the PESTANAL Atrazine ELISA Kit; Riedel deHaën) are pipetted into each well of a 96 well MultiScreen filter plate(PVDF membrane; Millipore GmbH, Eschborn, Germany) including 200 μl ofmicroparticle suspension (100 mg/ml) and incubated with agitation atroom temperature for 2 hours. This is followed by sucking off, washing,developing and quenching according to the protocol of the commercialassay (see above). The solutions are removed, and the absorbances at 450nm are measured in a UV spectrometer; measurements on particles modifiedwithout template (MTP-Blank) and determinations using another herbicideare used as controls (see Table 8).

[0097] In a solid-phase assay relating to the analytics of herbicides inan ELISA formate and according to an established ELISA protocol, the newTTP materials show pronounced specificity (TTP vs. blank) andselectivity (Atrazine template vs. Metribuzin non-template). TABLE 8Results of a competitive assay for Atrazine using Atrazine-peroxidaseconjugate and developer solutions (POD Assay) from a commercial AtrazineELISA kit with Atrazine-textured (MP-TTP) and control (MP-Blank)microparticles Absorbance (450 nm) Concentration 10⁻⁵ nM AtrazineMetribuzin MTP-TTP 0.75 0.81 MTP-Blank 0.86 0.85

[0098] The figures illustrate the following:

[0099] FIG. 1: Schematic illustration of the principles relating to thesynthesis and function of template-textured polymers (TTP) according tothe prior art.

[0100] FIG. 2: Schematic illustration of the production of TTP compositematerials with high specificity and selectivity by surfacefunctionalization of special supports:

[0101] a) Two-layer structure of a solid, mechanically stable support ofany morphology: the support material has a thin polymer layer thereonwhich minimizes non-specific binding of substances to the support.

[0102] b) The reaction mixture for TTP synthesis, including template andfunctional monomer, and optionally initiator, crosslinker and solvent,is placed on the thin polymer layer, penetrating same but not thesupport material. Compared to the free monomer and the other componentsof the reaction mixture, the complex of template and functional monomercan be subject to hindrance by the thin polymer layer (e.g. “sizeexclusion effect”), which may result in an accumulation oftemplate-complexed functional monomer on the exterior surface of thesupport. In control preparations with no template (and thus, with nocomplex), such effects are absent.

[0103] c) Crosslinking polymerization is initiated selectively on theboundary surface of support material/thin polymer layer or/and withinthe thin polymer layer; therefore, functionalization exclusively takesplace within the thin polymer layer. The functional groups of thecomplexed functional monomer are fixed as “template impressions”preferably on the exterior surface of the support. In controlpreparations with no template, there is no accumulation of functionalmonomer on the exterior surface (cf., b)) and consequently, specificbinding is absent and non-specific binding is exceedingly low. Incontrast, the TTP composite material has high binding specificity andselectivity for the template (owing to the “template impressions”), withlow non-specific binding (owing to the properties of the thin polymerlayer).

[0104] FIG. 3: Schematic illustration of the potential uses of TTPcomposite materials with high specificity and selectivity, exemplifiedby membranes for substance-specific affinity separation and/oranalytics.

[0105] FIG. 4: Terbumeton sorption (from water) in filtration (cf.,Example 2) for hydrophilized PVDF membranes following syntheses with(TTP) and without (blank) the Terbumeton template—variation offunctional monomer (cf., Example 1a).

[0106] FIG. 5: Terbumeton sorption (from water) in filtration (cf.,Example 2) for hydrophilized PVDF membranes following syntheses with(TTP) and without (blank) the Terbumeton template—variation offunctional monomer concentration (cf., Example 1b).

[0107] FIG. 6: Terbumeton sorption (from water) in filtration (cf.,Example 2) for hydrophilized PVDF membranes following syntheses with(TTP) and without (blank) the Terbumeton template—variation ofcrosslinker concentration (cf., Example 1c).

[0108] FIG. 7: Herbicide sorption (from water) in filtration (cf.,Example 2) for hydrophilized PVDF membranes following syntheses with(TTP) and without (blank) the Terbumeton template−variation offunctional monomer concentration (cf., Example 1a).

ABBREVIATIONS:

[0109] AA Acrylic acid

[0110] AMPS 2-Acryloylamino-propane-2-sulfonic acid

[0111] BET Brunauer-Emmet-Teller; method of measuring adsorptionisotherms to determine the specific surface area of solids

[0112] BP Benzophenone

[0113] DM Degree of Modification

[0114] ELISA Enzyme-linked immunosorbent assay

[0115] FT-IR-ATR Fourier Transform Infrared-Attenuation of TotalReflexion

[0116] GC Gas chromatography

[0117] S Solvent

[0118] M mol/l; concentration

[0119] MAA Methacrylic acid

[0120] MBAA n,n′-Methylenebisacrylamide

[0121] mM mol/l; concentration

[0122] MTP Microtiter plate

[0123] MP Microparticle

[0124] n Quantity of substance

[0125] POD Peroxidase

[0126] PP Polypropylene

[0127] PVDF Poly(vinylidene fluoride)

[0128] SEM Scanning electron microscopy

[0129] TTP Template-textured polymers

[0130] UV Ultraviolet

1. A method of producing a template-textured material by synthesis of atemplate-textured polymer (TTP) using crosslinking polymerization offunctional monomers in the presence of a template on a support,characterized in that a support having a thin polymer layer on thesurface thereof is added with a reaction mixture consisting ofpolymerization initiator, template, functional monomer, crosslinkingagent, solvent and/or buffer and, following sorption of the reactionmixture in the thin polymer layer, the polymerization is initiated andcontinued until the absorption capacity of the thin polymer layer forthe template-textured polymer is reached, and the template is optionallyremoved in a final step, the support used being selected in such a waythat it cannot absorb the reaction solution.
 2. The method according toclaim 1, characterized in that the reaction mixture is added in such away that initially, the major portion of the polymerization initiator ina solvent is added and subsequently, the reaction mixture comprised ofinitiator residual amount, template, functional monomer, crosslinker,solvent and/or buffer is added.
 3. The method according to claim 1,characterized in that the reaction mixture is added in such a way thatinitially, the entire polymerization initiator in a solvent is added andsubsequently, the residual reaction mixture comprised of template,functional monomer, crosslinker, solvent and/or buffer is added.
 4. Themethod according to claim 1, characterized in that a molded piece madeof a hydrophobic material is used as support, and a layer of ahydrophilic polymer is used as thin polymer layer.
 5. The methodaccording to claim 1, characterized in that an inorganic solid is usedas support, and a layer of a hydrophilic or hydrophobic polymer is usedas thin polymer layer.
 6. The method according to claim 1, characterizedin that a molded piece comprised of a highly crosslinked organic polymeris used as support, and a layer of a hydrophilic or hydrophobic polymeris used as thin polymer layer.
 7. The method according to any of claims4, 5 or 6, characterized in that films, foils, plates, particularlymicrotiter plates, reaction vessels of any shape, particles,particularly micro- or nanoparticles, fibers, particularly hollowfibers, fabrics, fleeces, filters, or membranes made of inorganic ororganic materials are used as support.
 8. The method according to claim4, characterized in that hydrophobic organic polymers are used ashydrophobic support materials, preferably polypropylene, polyethylene,polystyrene, polysulfone, hydrophobic polyamides, hydrophobicpolyesters, polycarbonate, polyacrylonitrile, poly(vinylidene fluoride),polytetrafluoroethylene, hydrophobic polyacrylates, as well asderivatives, copolymers or blends of these polymers.
 9. The methodaccording to claim 5, characterized in that glasses, silicates,ceramics, or metals or composites thereof, also including hydrophobic orcrosslinked organic polymers, are used as inorganic solids.
 10. Themethod according to claim 6, characterized in that highly crosslinkedpolystyrene and polystyrene derivatives or copolymers, or highlycrosslinked polyacrylates are used as highly crosslinked organicpolymer.
 11. The method according to claim 7, characterized in that aporous membrane having a pore size between 2 nm and 10 μm, preferablyfrom 100 nm to 5 μm, is used as support.
 12. The method according to anyof claims 4, 5 or 6, characterized in that a layer of crosslinked ornon-crosslinked hydrophilic polyacrylates, polyacrylamides, cellulose,amylose, agarose, as well as derivatives, copolymers or blends thereof,particularly of 2-hydroxypropyl methacrylate crosslinked withtetraethylene glycol bismethacrylate, is used as thin hydrophilicpolymer layer.
 13. The method according to claim 5 or 6, characterizedin that a layer of crosslinked or non-crosslinked fluorinated polymers,silicones, paraffins, or waxes, as well as derivatives, copolymers orblends thereof is used as thin hydrophobic polymer layer.
 14. The methodaccording to claim 1, characterized in that the thickness of the polymerlayer on the support is from 1 nm to 1 μm, preferably about 5 to 20 nm.15. The method according to claim 4, characterized in that ahydrophilized poly(vinylidene fluoride) membrane is used as hydrophobicsupport having a thin hydrophilic polymer layer on the surface thereof.16. The method according to claim 1, characterized in that thepolymerization is conducted as a photoinitiated crosslinking graftcopolymerization of the functional monomers, using an H abstraction typesubstance as photoinitiator and the thin polymer layer as co-initiator.17. The method according to claim 1, characterized in that smallmolecules having a molecular mass of up to 100 Da, larger molecules ofup to 1,000,000 Da, or even microorganisms or cells are used astemplates.
 18. The method according to claim 1, characterized in thatpolymerizable compounds including groups capable of interacting withtemplates, particularly carboxyl, sulfonyl, sulfate, phosphate, amino,or quaternary ammonium groups, or derivatives thereof, also inadmixture, preferably AMPS, MAA or AA, are used as functional monomers.19. A template-textured material, produced according to the method asclaimed in claims 1 to
 18. 20. Use of the template-textured materialaccording to claim 19 in the separation of materials or in analytics offluid or gaseous mixtures of substances, which procedures are based onspecific binding of the template or template derivative to TTP duringperfusion or diffusion through the template-textured material or whenapplied onto the template-textured material.
 21. The use according toclaim 20 in substance-specific separation of materials by means ofaffinity filtration using a TTP material according to claim 19 to effectconcentration, purification, separation, liberation, or analyticaldetermination of substances.
 22. The use according to claim 20 insubstance-specific separation of materials by means of dialysis orelectrodialysis using a TTP material according to claim 19 to effectconcentration, purification, separation, delivery, or analyticaldetermination of substances.
 23. The use according to claim 20 insubstance-specific separation of materials by means of solid-phaseextraction, chromatography, membrane chromatography, electrophoresis, orcontrolled liberation from a reservoir using a TTP material according toclaim 19 to effect concentration, purification, separation, delivery, oranalytical determination of substances.
 24. The use according to claim20 in substance-specific separation of materials and/or binding and/orchemical conversion by means of a TTP material according to claim 19 assensor or catalyst to effect purification, separation or analyticaldetermination of substances.
 25. The use according to claim 20 insubstance-specific separation of materials and/or binding and/orchemical conversion by means of a TTP material according to claim 19 asblotting membrane, test strip or support, preferably as reaction vessel,microtiter plate or particles for qualitative or quantitative assays orin active substance screening.